Amino acids fluorescamine deriv

Figure 4.19. Fluorescent Derivatives of Amino Acids. Fluorescamine reacts with the a -amino group of an amino acid to form a fluorescent derivative.

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

The use of chemiluminescence reactions for the detection of metal ions by liquid chromatography was recently reported [59,60]. The detectors made use of the chemiluminescence produced in the reaction between luminol and hydrogen peroxide which is catalyzed by transition metals. The column effluent was mixed with the reagents in order to yield the chemiluminescence. The reaction was fast and was carried out at room temperature. By varying the pH of the buffer, selectivity towards certain metals was also achieved. For example, at pH 10-11 nickel could be analyzed but lead and aluminium were inactive at pH 13-14, the converse was true [59]. Aminco-Bowman has marketed a liquid chromatographic system in which amino acids and amines are analyzed by means of the fluorescence produced on reaction with the reagent fluorescamine. Fluorescamine does not fluoresce, but it does react with primary amino groups to produce fluorescent derivatives. The reaction is instantaneous and may be carried out at room temperature, usually at pH 9. This detection system promises to be far more sensitive than the ninhydrin detection system and is much more easily adapted to HPLC. 

Since fluorescamine reacts only with primary amino groups, secondary amino acids do not give a fluorescent product with this reaction. A method for converting secondary amino acids into primary amines has been described for analysis using fluorescamine [87], and is based on treatment of the amino acid with N-chlorosuccinimide. The reaction involves an oxidative decarboxylation of the amino acids. This method has been incorporated into the automatic analysis of amino acids with fluorescamine [88]. The fluorescence spectra and the sensitivities are similar to those of the derivatives of the primary amino acids. 

Fluorescamine (4-phenylspiro(furan-2-(3H),r-phthalan)3,3 -dione) is also a commonly used fluorescence reagent. It reacts almost instantly and selectively with primary amines, while the excess of the reagent is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction is carried out in aqueous acetone at a pH of about 8-9 and the derivatives can be chromatographed directly. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Two disadvantages of the reagent are its cost and the fact the products are less stable, cannot be stored and should be injected onto the column immediately after formation. Fluorescamine has been employed in the analysis of polyamines, catecholamines and amino acids. 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

Their relative abundance can be determined by first treating the amino acids with ninhydrin (see Fig. 3-56) to create a colored derivative, or fluorescamine to generate a fluorescent derivative. The concentration of each amino acid is proportional to the absorbance (or fluorescence) of the solution. The technique was originally used to help identify proteins, but it is now more commonly used as the most accurate way to determine the concentration of a protein sample. 

Fluorescamine (50) reacts even more readily with a-amino acids [Eq. (8)]. The spectral characteristics are similar to those of MDPF derivatives. The CD spectra of the in situ reaction mixtures (20 different amino acids investigated) show three or four Cotton effects between 400 and 220 nm. As with the MDPF derivatives, the first Cotton effects (396-377 nm) of the chromophores derived from L-amino acids and fluorescamine are positive, and the second Cotton effects are negative. Within the experimental error, the CD curves of the chromophores derived from D-amino acids are mirror... 

Table III. Ultraviolet and CD Spectral Data for MDPF and Fluorescamine Derivatives of a-Amino Acids... 

On the basis of these results, it is apparent that the derivation of the absolute configuration of a COOH- or NH2-terminal amino acid in a dipeptide from its chiroptical properties is hazardous, and a comparison of spectral data with those of proper model peptides is needed. Thus, a more reliable technique is to react the NH2-terminal (or COOH-terminal) amino acid of a small peptide with a proper chromophoric reagent, as discussed above (via Edman degradation or other techniques). Cyclic peptides have to undergo at least partial acid hydrolysis before they can be reacted with methyl isothiocyanate, fluorescamine, etc. 

Fluorescamine under the alkaline conditions rapidly reacts with primary amines and amino acids to give fluorescent derivatives at room temperature. The advantageous features of this reaction are as follows (1) fluorescamine is nonfluorescent (2) fluorescamine can be hydrolyzed to the nonfluorescent product (3) the reaction with secondary amines can form nonfluorescent derivatives, which allows selectivity to primary amines. For those reasons, fluorescamine can be applied to pre- and postcolumn derivatization of primary amino compounds with LC-FL or CZE-LIF detection. 

The derivatives are used for amino acid analysis via HPLC separation. Instead of mercapto-ethanol, a chiral thiol, e.g., N-isobutyryl-L-cysteine, is used for the detection of D-amino acids. The detection hmit lies at 1 pmol. The very fast racemizing aspartic acid is an especially suitable marker. One disadvantage of the method is that proline and hydroxyproline are not detected. This method is apphed, e.g., in the analysis of fruit juices, in which high concentrations of D-amino acids indicate bacterial contamination or the use of highly concentrated juices. Conversely, too low concentrations of D-amino acids in fermented foods (cheese, soy and fish sauces, wine vinegar) indicate unfermented imitations. Fluorescamine reacts with primary amines and amino acids - at room temperature under alkaline conditions - to form fluorescent pyrrolidones (Aex = 390 nm, Aem - 474 nm). The detection limit lies at 50-100 pmol ... 

Figure 11 Determination of panthenol in multivitamin tablets. Chromatographic conditions see Table 3. Peak identification (1) 6-aminohexanoic acid (internal standard)-fiuo-rescamine derivative (2) 3-amino-1-propanol fluorescamine derivative. (From Ref. 67.)...

Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),l -phthalan]-3,3 -dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compoimds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm. 

In the first step tin(Il) chloride in acetic acid solution reduces the aromatic nitro groups to amino groups. The aromatic amines produced then react with fluorescamine in weakly basic medium to yield fluorescent derivatives (cf. reagent monograph Fluorescamine Reagent , Volume la). 

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